The overall goal of this application is to develop a quantitative in-vivo small animal imaging system for fluorescent reporter probes that fuses fluorescence light emitting computed tomography (FLECT) with X- ray computed tomography (CT). The proposed dual-modality imaging system will not only provide a research tool for better understanding of biological function and processes on a cellular or molecular level in-vivo, but will also aid the development of new drug therapies and accelerate their translation into the clinic. Conventional imaging methods only provide two-dimensional (2D) fluorescence surface images and, hence, do not reveal the actual spatial location and concentration of the targeted reporter system. Furthermore, current fluorescence tomography (FT) systems are still in a developing stage and suffer from several limitations. First, these FT systems assume optically uniform tissue models that, consequently, prohibit the accurate quantification of the reporter probe's location and concentration. Second, neither planar fluorescence imaging nor FT provides any anatomical information. Hence, the reconstructed reporter probe location cannot be localized relative to the animal's anatomy. The proposed FLECT/CT system will overcome these limitations in two ways. First, we will leverage the anatomical information gained from CT with its high spatial resolution and assign optical properties to various segmented organs. These non-uniform optical property maps will in turn improve quantitative fluorescence image reconstruction leading to accurate images about the reporter probe's actual spatial location and concentration. Second, structural images from CT will provide the anatomical information that is necessary for co-locating the fluorescent reporter probe to the animal's anatomy. In Phase 1, we will perform numerical simulations and tissue phantom experiments that will provide a proof of principle for the proposed FLECT/CT system. We will demonstrate that (1) applying non-uniform optical property maps to FLECT reconstructions makes quantitative tomographic imaging of reporter probes feasible and (2) spatial maps of organs with largely varying optical properties can be segmented from CT images. In Phase 2, a commercial grade FLECT/CT system will be developed where the optical and X-ray components share the same rotating gantry. We will develop fully automated image segmentation methods and different techniques for assigning optical parameters to segmented organs. The optical parameters will be determined by (1) optical tomography in a reduced parameter space, (2) from known (oxy-)hemoglobin concentrations in different tissue types, or from (3) optical parameter databases of prior experiments. Last, the performance of the FLECT/CT system will be evaluated in small animal imaging experiments. Once completed, our FLECT/CT system will provide a powerful tool for research of cancer, neurological pathologies, and cardiovascular disease. PUBLIC HEALTH RELEVANCE: The proposed development of a combined fluorescence tomography and X-ray CT imaging system for small animals will reconstruct and display the three-dimensional in-vivo distribution of fluorescent reporter probes for studying molecular processes in a living biological system. The combination of fluorescence tomography with X-ray CT will significantly improve the image quality of fluorescence tomographic images and will co-register them to structural CT images showing the animal's anatomy. Therefore, the proposed imaging system would not only be of great significance for better understanding biological processes and pathological function in living small animals on a cellular and molecular level, but would also aid the development of new drug therapies and accelerate their translation into the clinic.